Within this application several publications are referenced by Arabic numberals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
A low affinity receptor for IgG immune complexes, FcγRIII(CD16), is expressed on human natural killer (NK) cells and macrophages as an integral membrane glycoprotein anchored through a transmembrane peptide; on polymorphonuclear neutrophils (PMN) the receptor is anchored through a phosphatidylinositol (PI) linkage. The protein on NK cells macrophages has a molecular mass 6–10 kDa larger than that on PMN, and, unlike the latter, is resistant to PI-specific phospholipase C (PI-PLC). FcγRIII(CD16) transcripts isolated from PMN and NK cells of single donors revealed multiple single nucleotide differences, one of which converts an in frame UGA termination codon to CGA codon. The resulting open reading frame encodes a longer cytoplasmic domain for FcγRIII(CD16) in NK cells, contributing to its transmembrane anchor. Two nearly identical, linked genes which encode these transcripts have been cloned for FcγRIII(CD16), one of which (III-1) is allelic for NA-1 and NA-2. The allelic sites have been mapped to two single nucleotides in the extracellular domain. These genes are transcribed in a cell-type specific fashion to generate the alternatively anchored forms of this receptor.
Receptors for the Fc fragment of immunglobulin G (FcγR) couple the humoral and cellular immune responses by targetting immune complexes to effector cells. Multiple FcγRs exist, which differ in ligand affinity, cellular distribution and effector function (reviewed in 1). Detailed characterization of the FcγRs in both mouse and human has begun to address the molecular basis for the diversity of cellular responses triggered by a common ligand. The binding of immune complexes is mediated by extracellular immunoglobulin-like domains which are conserved among many FcγRs. The functional consequence of this binding, on the other hand, is mediated by the divergent transmembrane and cytoplasmic domains which are the result of gene duplication as well as alternative mRNA splicing. In the mouse the low affinity, immune complex IgG Fc receptors (FcγRII) are encoded by two genes, α and β (2, 3, 4). cDNA sequence analysis predicts that these receptors are similar integral membrane glycoproteins with 180 amino acid extracellular domains, single transmembrane spanning domains of 20 amino acids and intracytoplasmic domains which very from 26 amino acids for α to 93 amino acids for the larger spliced form of β, β1. α is expressed on macrophages and NK cells, while β is expressed on lymphocytes and macrophages and displays cell type-specific alternative mRNA splicing of its cytoplasmic domains. The human homologues of these receptors include a minimum of three genes for FcγRII (CD32) (Ravetch, et al. unpublished) and two genes for FcγRIII(CD16) (this report). cDNA clones have been isolated for FcγRII (CD32) (5,6, Ravetch, et al. unpublished) and FcγRIII(CD16) (7,8).
FcγRIII(CD16) is expressed on NK cells, macrophages and PMN (9,10,11). Two alleles, NA-1 and NA-2, have been described for this receptor on PMN. It has been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) by NK cells (12) where it represents the only FcγR. On PMN, FcγRIII(CD16) has been proposed to act together with FcγRII (CD32) to mediate effector functions (13, 14). Anti-FcγRIII(CD16) antibodies inhibit ADCC and immune complex binding (9,10,15) on both PMN and NK cells. Recently it has been shown that FcγRIII(CD16) is anchored through a glycosyl-phosphatidylinositol (PI) linkage on PMN (13,16). The first evidence for an alternative membrane-associated form for FcγRIII(CD16) came from the study of patients with paroxysmal nocturnal hemoglobinuria (PNH). In that acquired disorder, a defect in the attachment of the PI tail in hematopoietic precursor cells results in the selective deficiency of PI-anchored proteins (reviewed in 17). FcγRIII(CD16) is expressed at 10% of normal levels on PMN in those patients, but its expression on macrophages and NK cells is unaffected (16,18), indicating that NK cells and macrophages express an alternative anchored form of FcγRIII(CD16) which is presumably transmembrane. cDNA clones for FcγRIII(CD16) have been isolated from placental and neutrophil libraries (7,8). Those clones predict a sequence for an FcγRIII(CD16) protein which contains two canonical immunoglobulin-like extracellular domains, a weakly hydrophobic transmembrane domain and a short (4 amino acid) cytoplasmic domain, features characteristic of PI-linked molecules (17,19). Transfection of those clones resulted in the appearance of PI-linked molecules on COS cells; thus it appeared unlikely that these cDNA clones encoded a transmembrane form of FcγRIII(CD16).
It has been shown that antibody-dependent enhancement (ADE) of infection by human immunodeficiency virus (HIV) of macrophages is mediated by FcγRIII(CD16) (40). In the presence of enhancing antibodies, HIV can infect a cell independently of the CD4 protein. The FcγRIII(CD16) receptor mediates uptake of HIV-enhancing antibody complexes into human macrophages.
The inventors have demonstrated that the FcγRIII(CD16) molecule indeed exists in two alternative membrane-anchored forms—a PI-linked form on PMN and a larger PI-PLC resistant transmembrane protein on NK cells. To establish the molecular basis for this difference, FcγRIII(CD16) encoding RNA from NK cells and PMN of single individuals homozygous for either NA-1 or NA-2 were analyzed and found to differ by multiple single nucleotide substitutions. One of these non-allelic changes results in the expression of a transcript in NK cells in which a CGA codon replaces a UGA termination codon thereby extending the reading frame for the cytoplasmic domain of this FcγRIII(CD16) by 21 amino acids, which are homologous to the murine FcγRIIα cytoplasmic domain. Two distinct genes encoding FcγRIII(CD16) have been cloned and sequenced. Cell-type specific expression of these linked genes accounts for the NK cell and PMN transcripts and the alternatively-anchored forms of this receptor.